Stains combine chemically with the bacterial protoplasm; if the cell is not already dead, the staining process itself will kill it. The process is thus a drastic one and may produce artifacts.

The commonly used stains are salts. Basic stains con sist of a colored cation with a colorless anion (eg, methylene blue+ chloride−); acidic stains are the reverse (eg, sodium+ eosinate−). Bacterial cells are rich in nucleic acid, bearing negatively charged phosphate groups. These combine with the positively charged basic dyes. Acidic dyes do not stain bacterial cells and hence can be used to stain background material a contrasting color (see Negative Staining).

The basic dyes stain bacterial cells uniformly unless the cytoplasmic RNA is destroyed first. Special staining techniques can be used, however, to differentiate flagella, capsules, cell walls, cell membranes, granules, nucleoids, and spores.

The Gram Stain

 An important taxonomic characteristic of bacteria is their response to Gram-stain. The Gram-staining property appears to be a fundamental one because the Gram reaction is correlated with many other morphologic properties in phylogenetically related forms. An organism that is potentially Gram-positive may appear so only under a particular set of environmental conditions and in a young culture.

The Gram-staining procedure begins with the application of a basic dye, crystal violet. A solution of iodine is then applied, which forms a complex with crystal violet. All bacteria will be stained blue at this point in the procedure. The cells are then treated with alcohol. Gram-positive cells retain the crystal violet–iodine complex, remaining blue; Gram-negative cells are completely decolorized by alcohol. As a last step, a counterstain (eg, the red dye safranin) is applied so that the decolorized Gram-negative cells will take on a contrasting color; the Gram-positive cells now appear purple (Table 1).

Table1. Comparison of Features of Gram-Positive and Gram-Negative Bacteria

The basis of the differential Gram reaction is the structure of the cell wall, as discussed earlier in this chapter.

The Acid-Fast Stain

Acid-fast bacteria are those that retain carbolfuchsin (basic fuchsin dissolved in a phenol–alcohol–water mixture) even when decolorized with hydrochloric acid in alcohol. A smear of cells on a slide is flooded with carbolfuchsin and heated on a steam bath. After this, the decolorization step with acid-alcohol is carried out, and finally a contrasting (blue or green) counterstain is applied. Acid-fast bacteria (mycobacteria and some of the related actinomycetes) appear red; others take on the color of the counterstain.

Negative Staining

This procedure involves staining the background with an acidic dye, leaving the cells contrastingly colorless. The black dye nigrosin is commonly used. This method is used for cells or structures that are difficult to stain directly.

The Flagella Stain

Flagella are too fine ( ca 20 nm in diameter) to be visible in the light microscope. However, their presence and arrangement can be demonstrated by treating the cells with an unstable colloidal suspension of tannic acid salts, causing a heavy precipitate to form on the cell walls and flagella. In this manner, the apparent diameter of the flagella is increased to such an extent that subsequent staining with basic fuchsin makes the flagella visible in the light microscope. Figure 1 shows cells stained by this method.

Fig1. Flagella stain of Pseudomonas species. (Reproduced with permission from Leifson E: Staining, shape and arrangement of bacterial flagella. J Bacteriol 1951;62:377.)

In peritrichous bacteria, the flagella form into bundles during movement, and such bundles may be thick enough to be observed on living cells by dark-field or phase-contrast microscopy.

The Capsule Stain

Capsules are usually demonstrated by the negative staining procedure or a modification of it. One such “capsule stain” (Welch method) involves treatment with hot crystal violet solution followed by a rinsing with copper sulfate solution. The latter is used to remove excess stain because the conventional washing with water would dissolve the capsule. The copper salt also gives color to the background, with the result that the cell and background appear dark blue and the capsule a much paler blue.

Staining of Nucleoids

 Nucleoids are stainable with the Feulgen stain, which is specific for DNA. The DNA-intercalating stains DAPI and ethidium bromide are widely used for fluorescence micros copy of nucleoids.

The Spore Stain

Spores are most simply observed as intracellular refractile bodies in unstained cell suspensions or as color less areas in cells stained by conventional methods. The spore wall is relatively impermeable, but dyes can be made to penetrate it by heating the preparation. The same impermeability then serves to prevent decolorization of the spore by a period of alcohol treatment sufficient to decolorize vegetative cells. The latter can finally be counterstained. Spores are commonly stained with malachite green or carbolfuchsin (Figure 2).

Fig2. Endospore stain. Endospores retain the green primary stain, malachite green. Counterstaining with safranin imparts a red color to other cells. (Courtesy of Larry Stauffer, Oregon State Public Health Laboratory. Source: Centers for Disease Control and Prevention, Public Health Image Library, ID# 1895, 2002.)

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مواضيع ذات صلة

Morphologic Changes During Growth

Endospores

The Acid-Fast Cell Wall

Special Components of Gram-Negative Cell Walls

Special Components of Gram-Positive Cell Walls

The Peptidoglycan Layer

Bacterial Cultivation

Principles of Bacterial Cultivation : Environmental Requirements

Structure and Function of the Bacterial Cell

Bacterial Metabolism

Bacterial Genetics: Nucleic Acid Structure and Organization

Detection of Bacteremia: Culture Techniques